Estimation of mean erythrocyte age using HbA1c or HbA1c/glycated albumin for evaluation of anemia severity.

BACKGROUND: Hemoglobin A1c (HbA1c) levels are low in patients with hemolytic anemia, as HbA1c reflects mean erythrocyte age (MRBC ). Erythrocyte creatine (EC) is a hemolytic indicator that also reflects MRBC . We previously reported an equation for estimating MRBC using EC (EC-MRBC ). AIMS: In this study, EC-MRBC was compared to the HbA1c level expressed in the International Federation of Clinical Chemistry and Laboratory Medicine units (iA1c) and to the iA1c/glycated albumin (GA) ratio to estimate MRBC . METHODS: This study included 238 subjects, including patients with hemolytic anemia and/or type 2 diabetes mellitus (T2DM). RESULTS: In non-diabetic individuals, both iA1c and iA1c/GA showed a strong positive correlation with EC-MRBC (p < 0.0001). The equations to estimate iA1c-MRBC and iA1c/GA-MRBC derived from the regression equations between EC-MRBC and iA1c, and EC-MRBC and iA1c/GA in nondiabetic individuals were 1.45 × iA1c and 20.0 × iA1c/GA, respectively. iA1c-MRBC and iA1c/GA-MRBC in non-diabetic individuals without hemolytic anemia were 57.6 ± 4.0 and 57.1 ± 6.4 days, respectively, and iA1c/GA-MRBC in T2DM patients without hemolytic anemia was 56.0 ± 8.8 days.; no significant difference was seen in the comparisons. CONCLUSIONS: The MRBC can be estimated using iA1c or iA1c/GA in non-diabetic individuals, and iA1c/GA in T2DM patients.

Usefulness of HbA1c Measurements by HPLC- and Immuno-Assay for Screening Unstable Hemoglobinopathy.

OBJECTIVE: Unstable hemoglobinopathy (UH), red blood cell membrane disease (MD), and red blood cell enzymopathy are known as major congenital hemolytic anemias. Specialized examinations are needed for their differential diagnosis. We hypothesized that simultaneous measurements of HbA1c levels using high-performance liquid chromatography (HPLC) by fast mode (FM) and immunoassay [HPLC (FM)-HbA1c and IA-HbA1c, respectively] are useful for the differential diagnosis of UH from other congenital hemolytic anemias and verified this hypothesis in this study. METHODS: HPLC (FM)-HbA1c and IA-HbA1c levels were simultaneously measured in 5 variant hemoglobinopathy (VH) patients with ß-chain heterozygous mutation, 8 MD patients, 6 UH patients, and 10 healthy controls. None of the patients had diabetes mellitus. RESULTS: In VH patients, HPLC-HbA1c levels were low, whereas IA-HbA1c levels were within the reference range. In MD patients, HPLC-HbA1c and IA-HbA1c levels were similarly low. In UH patients, both HPLC-HbA1c and IA-HbA1c levels were low, but HPLC-HbA1c levels were significantly lower than IA-HbA1c levels. The HPLC-HbA1c/IA-HbA1c ratio was 90% or more in all MD patients and control subjects. This ratio was, however, less than 90% in all VH patients and UH patients. CONCLUSION: The HPLC (FM)-HbA1c/IA-HbA1c ratio calculated using simultaneous measurements of HPLC (FM)-HbA1c and IA-HbA1c levels is useful for the differential diagnosis of VH, MD, and UH.

Diagnostic accuracy and clinical usefulness of erythrocyte creatine content to predict the improvement of anaemia in patients receiving maintenance haemodialysis.

BACKGROUND: The improvement of anaemia over time by erythropoiesis stimulating agent (ESA) is associated with better survival in haemodialysis patients. We previously reported that erythrocyte creatine content, a marker of erythropoietic capacity, was a reliable marker to estimate the effectiveness of ESA. The aim of this study was to examine the accuracy and clinical usefulness of erythrocyte creatine content to predict the improvement of anaemia in haemodialysis patients. METHODS: ESA dose was fixed 3 months prior to the enrollment and was maintained throughout the study period. Erythrocyte creatine content and haematologic indices were measured at baseline in 92 patients receiving maintenance haemodialysis. Haemoglobin was also measured 3 months after. Improvement of anaemia was defined as ≥ 0.8 g/dL change in haemoglobin from baseline to 3 months. RESULTS: Erythrocyte creatine content was significantly higher in 32 patients with improvement of anaemia compared to 60 patients with no improvement of anaemia (2.47 ± 0.74 vs. 1.57 ± 0.49 µmol/gHb, P = 0.0001). When 9 variables (erythrocyte creatine content, ESA dose, reticulocyte, haptoglobin, haemoglobin at baseline, serum calcium, intact parathyroid hormone, transferrin saturation and serum ferritin) were used in the multivariate logistic regression analysis, erythrocyte creatine emerged as the most important variable associated with the improvement of anaemia (P = 0.0001). The optimal cut-off point of erythrocyte creatine content to detect the improvement of anaemia was 1.78 µmol/gHb (Area under the curve: 0.86). Sensitivity and specificity of erythrocyte creatine content to detect the improvement of anaemia were 90.6% and 83.3%. CONCLUSION: Erythrocyte creatine content is a reliable marker to predict the improvement of anaemia 3 months ahead in patients receiving maintenance haemodialysis.

Measurement of erythrocyte creatine might be useful for diagnosing latent hemolysis.

OBJECTIVES: Whereas HbA1c values are low relative to glycemia in patients with hemolytic anemia, including compensatory anemia, low HbA1c levels along with negative results for conventional hemolysis indicators have been reported in patients with latent hemolysis. Conversely, glycated albumin (GA) is a glycemic control indicator unaffected by hemolysis. Erythrocyte creatine (EC) is a hemolysis indicator that reflects the mean age of red blood cells (MRBC). We recently reported a formula for obtaining MRBC based on EC. The present study examined the usefulness of EC measurements and MRBC calculated with EC for diagnosing latent hemolysis. MATERIALS AND METHODS: Two patients with latent hemolysis and low HbA1c values relative to glycemia were investigated, while controls comprised 214 patients (including patients with hemolysis and/or type 2 diabetes mellitus). HbA1c was expressed in International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) units (iA1c). GA/iA1c ratios, reticulocyte counts, EC, and MRBC in patients with latent hemolysis were compared to non-hemolysis, compensatory hemolysis, and hemolytic anemia patients. RESULTS: Both reticulocyte counts and haptoglobin levels were within reference ranges in patients with latent hemolysis. GA/iA1c ratios and EC were higher than reference values in patients with latent hemolysis, and MRBC values were 41.6 and 48.4 days, respectively, shorter than the reference range (49.1-66.8 days). CONCLUSIONS: EC measurement and MRBC values calculated on the basis of EC might be useful for diagnosing latent hemolysis.

Evaluation of recombinant human erythropoietin responsiveness by measuring erythrocyte creatine content in haemodialysis patients.

BACKGROUND: One of the main causes of anaemia in patients with end-stage renal disease is relative deficiency in erythropoietin production. Eythropoiesis stimulating agent (ESA), a potent haematopoietic growth factor, is used to treat anaemia in haemodialysis patients. The effect of ESA is usually assessed by haematological indices such as red blood cell count, haemoglobin concentration and haematocrit, but erythrocyte indices do not provide information of the rapid change in erythropoietic activity. As erythrocyte creatine directly assess erythropoiesis, the aim of this study was to evaluate the effect of ESA in haemodialysis patients by measuring the erythrocyte creatine content. METHODS: ESA dose was fixed 3 months prior to the enrollment and was maintained throughout the entire study period. Erythrocyte creatine was measured with haematologic indices in 83 haemodialysis patients. Haemoglobin was also measured 3 months after. RESULTS: ESA dose (152.4 ± 62.9 vs. 82.2 ± 45.5 units/kg/week, P = 0.0001) and erythrocyte creatine (2.07 ± 0.73 vs. 1.60 ± 0.41 µmol/gHb, p = 0.0003) were significantly higher in 27 patients with haemoglobin <10 g/dL compared to 56 patients with haemoglobin ≥10 g/dL. There was a fair correlation between ESA dose and the concentration of creatine in the erythrocytes (r = 0.55, P < 0.0001). Increase in haemoglobin (>0.1 g/dL) was observed in 37 patients, whereas haemoglobin did not increase in 46 patients. Erythrocyte creatine levels were significantly higher in those patients with an increase in haemoglobin compared to those without (2.04 ± 0.64 vs. 1.52 ± 0.39 µmol/gHb, p < 0.0001). When 8 variables (ESA dose, erythropoietin resistance index, C-reactive protein, intact parathyroid hormone, iron supplementation, presence of anaemia, erythrocyte creatine and reticulocyte) were used in the multivariate logistic analysis, erythrocyte creatine levels emerged as the most important variable associated with increase in haemoglobin (Chi-square = 6.19, P = 0.01). CONCLUSION: Erythrocyte creatine, a useful marker of erythropoietic capacity, is a reliable marker to estimate ameliorative effectiveness of ESA in haemodialysis patients.

Estimation of the hemoglobin glycation rate constant.

In a previous study, a method of obtaining mean erythrocyte age ([Formula: see text]) from HbA1c and average plasma glucose (AG) was proposed. However, the true value of the hemoglobin glycation constant ([Formula: see text] dL/mg/day), required for this model has yet to be well characterized. Another study also proposed a method of deriving [Formula: see text] from erythrocyte creatine (EC). Utilizing these formulae, this study aimed to determine a more accurate estimate of [Formula: see text]. One hundred and seven subjects including 31 patients with hemolytic anemia and 76 subjects without anemia were included in this study. EC and HbA1c data were analyzed, and [Formula: see text] using HbA1c, AG and the newly-derived constant, [Formula: see text] were compared to [Formula: see text] using traditional [Formula: see text] in three patients whose data were taken from previous case studies. A value of [Formula: see text] dL/mg/day was determined for [Formula: see text]. [Formula: see text] using HbA1c, AG and [Formula: see text] were found to no be significantly different (paired t-test, [Formula: see text]) to [Formula: see text] using traditional [Formula: see text]. [Formula: see text] enables the estimation of [Formula: see text] from HbA1c and AG.

An iPSC-based neural model of sialidosis uncovers glycolytic impairment-causing presynaptic dysfunction and deregulation of Ca2+ dynamics.

Sialidosis is a neuropathic lysosomal storage disease caused by a deficiency in the NEU1 gene-encoding lysosomal neuraminidase and characterized by abnormal accumulation of undigested sialyl-oligoconjugates in systemic organs including brain. Although patients exhibit neurological symptoms, the underlying neuropathological mechanism remains unclear. Here, we generated induced pluripotent stem cells (iPSCs) from skin fibroblasts with sialidosis and induced the differentiation into neural progenitor cells (NPCs) and neurons. Sialidosis NPCs and neurons mimicked the disease-like phenotypes including reduced neuraminidase activity, accumulation of sialyl-oligoconjugates and lysosomal expansions. Functional analysis also revealed that sialidosis neurons displayed two distinct abnormalities, defective exocytotic glutamate release and augmented α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptor (AMPAR)-mediated Ca2+ influx. These abnormalities were restored by overexpression of the wild-type NEU1 gene, demonstrating causative role of neuraminidase deficiency in functional impairments of disease neurons. Comprehensive proteomics analysis revealed the significant reduction of SNARE proteins and glycolytic enzymes in synaptosomal fraction, with downregulation of ATP production. Bypassing the glycolysis by treatment of pyruvate, which is final metabolite of glycolysis pathway, improved both the synaptsomal ATP production and the exocytotic function. We also found that upregulation of AMPAR and L-type voltage dependent Ca2+ channel (VDCC) subunits in disease neurons, with the restoration of AMPAR-mediated Ca2+ over-load by treatment of antagonists for the AMPAR and L-type VDCC. Our present study provides new insights into both the neuronal pathophysiology and potential therapeutic strategy for sialidosis.

A generic assay for the identification of splicing variants that induce nonsense-mediated decay in Pompe disease.

DNA variants affecting mRNA expression and processing in genetic diseases are often missed or poorly characterized. We previously reported a generic assay to identify variants that affect mRNA expression and splicing in Pompe disease, a monogenic disorder caused by deficiency of acid α-glucosidase (GAA). However, this assay could miss mRNA that is subjected to degradation. Here, we inhibited mRNA degradation using cycloheximide and performed unbiased splicing analysis of all GAA exons using exon flanking RT-PCR and exon internal RT-qPCR. In four patients that were suspected of harboring splicing variants but for which aberrant splicing could not be detected in normally growing cells, we detected a total of 10 novel splicing events in cells treated with cycloheximide. In addition, we found that sequences of GAA introns 6 and 12 were naturally included in a subset of transcripts from patients and healthy controls, indicating inefficient canonical splicing. Identification of aberrant splicing caused by the common Asian variant c.546G>T allowed the development of an antisense oligonucleotide that promoted canonical GAA pre-mRNA splicing and elevated GAA enzymatic activity. Our results indicate that this extended generic splicing assay allows the detection of aberrant splicing in cases of mRNA degradation to enable functional analysis of unknown splicing variants and the development of targeted treatment options.

Shortened red blood cell age in patients with end-stage renal disease who were receiving haemodialysis: a cross-sectional study.

BACKGROUND: The causes of anaemia in patients with end-stage renal disease include a relative deficiency in erythropoietin production and complex clinical conditions. We aimed to investigate the underlying mechanisms of anaemia in patients with end-stage renal disease who were undergoing maintenance dialysis by measuring erythrocyte creatine levels. METHODS: In a cross-sectional study, we evaluated 69 patients with end-stage renal disease who were receiving haemodialysis (n = 55) or peritoneal dialysis (n = 14). Erythrocyte creatine level, a quantitative marker of mean red blood cell (RBC) age, was measured. RESULTS: The mean RBC age was significantly shorter in the haemodialysis group than in the peritoneal dialysis group (47.7 days vs. 59.8 days, p < 0.0001), although the haemoglobin levels were comparable between the groups. A Spearman correlation coefficient analysis revealed that shortened RBC age positively correlated with transferrin saturation (r = 0.54), ferritin level (r = 0.47), and haptoglobin level (r = 0.39) but inversely related with reticulocyte (r = - 0.36), weekly doses of erythropoiesis-stimulating agents (ESAs; r = - 0.62), erythropoietin resistance index (r = - 0.64), and intradialytic ultrafiltration rate (r = - 0.32). CONCLUSIONS: Shortened RBC age was observed in patients who were receiving maintenance haemodialysis and was associated with iron deficiency, greater haptoglobin consumption, higher ESA requirements, and poor erythropoietin responsiveness, as well as with greater intradialytic fluid extraction.

A novel method for calculating mean erythrocyte age using erythrocyte creatine.

Estimating the lifespan of erythrocytes is useful for the differential diagnosis of anemia. However, measuring the lifespan of erythrocytes was very difficult; therefore, it was seldom measured. Erythrocyte creatine (EC) decreases reflecting erythrocyte age. We developed a method to obtain mean erythrocyte age (MRBC) from EC.We reanalyzed the previously published data from 21 patients with hemolytic anemia, which included EC and the half-life of 51Cr.MRBC and loge EC showed excellent significant linearity (r = -0.9475, p < 0.001), proving that it could be treated as a mono-exponential relationship within the studied range (EC: 1.45 - 11.76 µmol/g Hb). We established an equation to obtain MRBC (days) from EC (µmol/g Hb): MRBC = -22.84loge EC + 65.83.This equation allowed calculation of MRBC based on EC which has practical applications such as the diagnosis of anemia.

Novel Drug Candidates Improve Ganglioside Accumulation and Neural Dysfunction in GM1 Gangliosidosis Models with Autophagy Activation.

GM1 gangliosidosis is a lysosomal storage disease caused by loss of lysosomal ß-galactosidase activity and characterized by progressive neurodegeneration due to massive accumulation of GM1 ganglioside in the brain. Here, we generated induced pluripotent stem cells (iPSCs) derived from patients with GM1 gangliosidosis, and the resultant neurons showed impaired neurotransmitter release as a presynaptic function and accumulation of GM1 ganglioside. Treatment of normal neurons with GM1 ganglioside also disturbed presynaptic function. A high-content drug-screening system was then established and identified two compounds as drug candidates for GM1 gangliosidosis. Treatment of the patient-derived neurons with the candidate agents activated autophagy pathways, reducing GM1 ganglioside accumulation in vitro and in vivo, and restoring the presynaptic dysfunction. Our findings thus demonstrated the potential value of patient-derived iPSC lines as cellular models of GM1 gangliosidosis and revealed two potential therapeutic agents for future clinical application.

Sample transport and/or storage can cause falsely low HbA1c levels in blood cells measured by enzymatic assay.

Although HbA1c measurement by enzymatic assay (EA-HbA1c) is widely used in health-screening settings in Japan, recent studies have suggested lower EA-HbA1c levels as compared with HbA1c levels measured by high-performance liquid chromatography (HPLC-HbA1c). Hypothesizing that falsely low levels of EA-HbA1c are attributable to hemolysis caused by sample transport and/or storage, we measured EA-HbA1c in blood cells and whole blood after sample transport and compared them with HPLC-HbA1c levels. Blood samples were collected from ten non-diabetic individuals into sodium fluoride-containing blood collection tubes and immediately measured for EA-HbA1c in blood cells. After transport, the blood samples were again subjected to measurement of EA-HbA1c levels in blood cells and whole blood the following day. These EA-HbA1c levels were compared with HPLC-HbA1c levels. EA-HbA1c levels in blood cells measured immediately after sample collection did not significantly differ from HPLC-HbA1c levels. Transported blood samples showed hemolysis and significantly lower EA-HbA1c levels in blood cells, as compared with HPLC-HbA1c levels, whereas no significant difference was observed between EA-HbA1c levels in whole blood and HPLC-HbA1c levels. Transported blood samples showed hemolysis and falsely low EA-HbA1c levels in blood cells. Hemolysis caused by sample transport and/or storage might be responsible for the falsely low EA-HbA1c levels. This should be kept in mind, because falsely low HbA1c levels may lead to a false-negative diagnosis of diabetes.

HbA1c levels measured by enzymatic assay during off-site health checkups are lower than those measured by on-site HPLC assay.

HbA1c is widely used as a therapeutic target marker and as a diagnostic marker for diabetes mellitus. This has led to an increasing frequency of HbA1c measurements in current health checkups throughout Japan. In the present study, we compared the HbA1c levels measured by an enzymatic assay (EA-HbA1c) off-site during health checkups with the HbA1c levels measured by on-site ion-exchange high-performance liquid chromatography (HPLC; HPLC-HbA1c) in a hospital. A total of 96 individuals (53 males and 43 females; age, 68.9 ± 8.4 years old; 70 diabetic and 26 non-diabetic individuals) whose HbA1c levels were measured by both the methods listed above were included in the study. Since no HPLC-HbA1c levels were measured on the day of the health checkup, HPLC-HbA1c levels were estimated using HPLC-HbA1c levels measured before and after the health checkup. A significant correlation of HbA1c levels was observed between the two groups (R = 0.973; p < 0.001). However, EA-HbA1c levels measured off-site during health checkups are lower than estimated HPLC-HbA1c levels measured on-site (6.37 ± 0.75% vs. 6.69 ± 0.75%; p < 0.001). Since lower EA-HbA1c levels measured during health checkups, which diverged from on-site measurements, may lead to underestimating diabetes mellitus, accurate measurement of HbA1c is required irrespective of the measuring method. Further investigation of the cause of falsely low EA-HbA1c levels and the strategy for reconciling HbA1c to reflect plasma glucose accurately are warranted.

HbA1c adjusted by erythrocyte creatine is a useful glycemic control indicator in patients with hemolysis.

OBJECTIVES: HbA1c shows low in patients with hemolysis, whereas glycated albumin (GA) is not affected by hemolysis. Therefore, the GA/HbA1c ratio reflects hemolysis in diabetic patients with hemolysis. Erythrocyte creatine (EC) is an indicator of hemolysis that reflects the mean erythrocyte age. The aim of this study was to examine whether HbA1c adjusted by EC accurately reflected glycemic control in patients with hemolysis. MATERIALS AND METHODS: A total of 238 individuals, consisting of 131 diabetic patients and 107 non-diabetic subjects, and consisting of 42 patients with hemolysis, and 196 subjects without hemolysis were selected for the study. HbA1c expressed in the IFCC units (iA1c) as well as in the NGSP units (A1C) were used. From the fact that EC and the GA/iA1c ratio showed a significant positive correlation, a formula for iA1c adjusted by EC (ECadj-iA1c) was created from a regression equation between EC and the GA/iA1c ratio. RESULTS: Significant correlations were observed between the GA/iA1c ratio and various hemolytic indicators but not between the GA/ECadj-iA1c ratio and those hemolytic indicators. The GA/iA1c ratio in individuals with hemolysis was significantly higher than in individuals without hemolysis, while no significant differences were observed in the GA/ECadj-iA1c ratio between the groups. Further, iA1c concentrations in non-diabetic patients with hemolysis were significantly lower than in the non-diabetic subjects without hemolysis, whereas ECadj-iA1c and GA concentrations showed no significant difference between the two groups. CONCLUSIONS: These results suggested that ECadj-iA1c accurately reflected glycemic control in patients with hemolysis.

Hemolysis causes a decrease in HbA1c level but not in glycated albumin or 1,5-anhydroglucitol level.

HbA1c has been widely used as a glycemic control indicator or as a diagnostic indicator for diabetes mellitus. However, HbA1c is affected by the erythrocyte life span and, therefore, shows falsely low values in hemolytic patients. Erythrocyte creatine (EC) is a sensitive hemolytic marker that reflects the mean erythrocyte age. In the present study, the relationships of HbA1c, glycated albumin (GA), and 1,5-anhydroglucitol (1,5-AG) with different hemolytic markers, including EC, were investigated in non-diabetic individuals. A total of 43 non-diabetic individuals whose complete blood count and reticulocytes were measured via medical examinations were included in this study (28 individuals with hemolysis and 15 individuals without hemolysis). Those with suspected diabetes mellitus based on medical history, low 1,5-AG values, or had comorbid liver and renal diseases were excluded from this study. HbA1c, GA, 1,5-AG, and various hemolytic markers were measured to examine the correlation of the glycemic control indicators with the various hemolytic markers. A significant correlation was observed between GA and 1,5-AG but not between HbA1c and GA or 1,5-AG. Significant correlations were observed between HbA1c values and various hemolytic markers (reticulocytes, haptoglobin, and EC) but not between GA or 1,5-AG values and those hemolytic markers. HbA1c, but not with GA and 1,5-AG, showed significant correlations with the hemolytic markers. These results suggested that HbA1c does not reflect the glycemic control accurately in hemolytic patients, while GA and 1,5-AG values are not affected by mean erythrocyte age and, therefore, accurately reflect the glycemic control.

Newborn screening for Pompe disease in Japan: report and literature review of mutations in the GAA gene in Japanese and Asian patients.

A newborn screening program for Pompe disease using dried blood spots (DBSs) was initiated in Japan. Here, we summarized this screening program and described the results of the GAA gene analysis. From April 2013 to November 2016, 103,204 newborns were screened; 71 had low acid alpha-glucosidase (AαGlu) activity. GAA sequencing showed that 32 (45.1%) and 37 (52.1%) of these newborns were homozygous and heterozygous for pseudodeficiency alleles c.[1726G>A; 2965G>A], respectively. Moreover, 24 of 32 newborns with homozygous c.[1726G>A; 2965G>A] alleles had no mutations, and the other eight had one mutation each. Thirty-five of 37 newborns with heterozygous c.[1726G>A; 2965G>A] alleles had one mutation, and the other two had two mutations each. Only one newborn who had two mutations did not harbor c.[1726G>A; 2965G>A] alleles. Thus, it was difficult to distinguish newborns with c.[1726G>A; 2965G>A] alleles from newborns with pre-symptomatic Pompe disease using AαGlu assays in DBSs or fibroblasts; GAA gene sequencing was necessary. Seventy-one newborns had 50 variants, including 21 mutations or predictably pathogenic variants, and 29 polymorphisms or predictably non-pathogenic variants. Four of 21 mutations or predictably pathogenic variants and four of 29 polymorphisms or predictably non-pathogenic variants were novel. No infantile-onset Pompe disease was detected, and three newborns were diagnosed with potential late-onset Pompe disease. In the literature, 156 variants have been reported for 296 patients from 277 families in 41 articles from Japan, Korea, Taiwan, and China. Our results provide insights into GAA gene mutation profiles and the relationship between GAA and Pompe disease in Asian populations.

Shortened mean erythrocyte age in female patients with type 2 diabetes mellitus.

BACKGROUND: The hyperglycemic state is known to shorten the erythrocyte life span. Erythrocyte creatine (EC) reflects the mean erythrocyte age and is useful as an indicator of hemolysis. Here, we studied the relationship between EC and glycemic control indicators [HbA1c or glycated albumin (GA)] in non-diabetic subjects and diabetic patients. METHODS: This study included 119 patients with type 2 diabetes mellitus (T2DM) and 76 non-diabetic subjects matched by sex and age. We studied the relationships between EC and HbA1c or GA in patients with T2DM and non-diabetic subjects. RESULTS: Erythrocyte creatine in T2DM patients was significantly higher than that in non-diabetic subjects, and the ratio of high EC levels (>1.8 µmol/g Hb) in T2DM patients was significantly higher as well. Furthermore, female EC was significantly higher than male EC, and the ratio of high EC levels in females was significantly higher than in the males as well. While male EC had no significant correlation with HbA1c or GA, female EC had significant positive correlations with both. Male EC had no significant difference between T2DM patients and non-diabetic subjects, while the EC in female patients with T2DM was significantly higher than in female non-diabetic subjects. CONCLUSIONS: The significant positive correlations of EC with HbA1c and GA in female patients with T2DM suggested that the mean erythrocyte age decreased in female diabetic patients with poor glycemic control.

Intravascular hemolysis in patients with mitral regurgitation: Evaluation by erythrocyte creatine.

BACKGROUND: Intravascular hemolysis has been reported in patients with cardiac valve prostheses, but intravascular hemolysis in patients with mitral regurgitation with native valve has not been evaluated in detail. We designed a study to elucidate the impact of regurgitation flow on intravascular hemolysis in patients with primary mitral regurgitation by measuring erythrocyte creatine. METHODS: Erythrocyte creatine was enzymatically assayed in 29 patients with moderate to severe primary mitral regurgitation and 12 age-matched healthy volunteers. The size and characteristics of mitral regurgitation were determined by color Doppler echocardiography. RESULTS: Erythrocyte creatine was significantly higher in patients with eccentric jet (n=17, 2.64±0.77µmol/g Hb) than that of central jet (n=12, 1.68±0.13µmol/g Hb) and control subjects (1.39±0.25µmol/g Hb). Patients with eccentric jet had a significantly lower erythrocyte count and hemoglobin (385±58 x104/µL and 116±19g/l) compared to those with central jet (450±47×104/µL and 137±14g/l) and control subjects (433±31×104/µL and 134±19g/l). There were no significant differences in age, estimated glomerular filtration rate, pulmonary artery systolic pressure, left atrial size and left ventricular end-diastolic dimension between patients with eccentric jet and central jet. CONCLUSIONS: Intravascular hemolysis associated with subclincal anemia in patients with eccentric jet was due to the destruction of erythrocyte by collision of the eccentric jet to the atrial wall.

Evaluation of Intravascular Hemolysis With Erythrocyte Creatine in Patients With Aortic Stenosis.

Chronic intravascular hemolysis has been identified in patients with cardiac valve prostheses, but only a few case reports have evaluated intravascular hemolysis in patients with native valvular heart disease. To detect intravascular hemolysis in patients with aortic stenosis, erythrocyte creatine was evaluated with hemodynamic indices obtained by echocardiography.Erythrocyte creatine, a marker of erythrocyte age, was assayed in 30 patients with aortic stenosis and 10 aged matched healthy volunteers. Peak flow velocity of the aortic valve was determined by continuous-wave Doppler echocardiography. Twenty of 30 patients with aortic stenosis had high erythrocyte creatine levels (> 1.8 µmol/g Hb) and erythrocyte creatine was significantly higher as compared with control subjects (1.98 ± 0.49 versus 1.52 ± 0.19 µmol/g Hb, P = 0.007). Peak transvalvular pressure gradient ranged from 46 to 142 mmHg and peak flow velocity ranged from 3.40 to 5.95 m/second. Patients with aortic stenosis had a significantly lower erythrocyte count (387 ± 40 versus 436 ± 42 × 10(4) µL, P = 0.002) and hemoglobin (119 ± 11 versus 135 ± 11 g/L, P < 0.001) as compared with control subjects. Erythrocyte creatine had a fair correlation with peak flow velocity (r = 0.55, P = 0.002).In conclusion, intravascular hemolysis due to destruction of erythrocytes was detected in patients with moderate to severe aortic stenosis and the severity of intravascular hemolysis was related to valvular flow velocity of the aortic valve.

Erythrocyte creatine as a marker of intravascular hemolysis due to left ventricular outflow tract obstruction in hypertrophic cardiomyopathy.

BACKGROUND: Erythrocyte creatine, a marker of erythrocyte age that increases with shortening of erythrocyte survival, has been reported to be a quantitative and reliable marker for intravascular hemolysis. We hypothesized that hemolysis could also occur due to intraventricular obstruction in patients with hypertrophic cardiomyopathy (HCM). The purpose of this study was to examine the presence of subclinical hemolysis and the relation between intravascular hemolysis and intraventricular pressure gradient (IVPG). METHODS AND RESULTS: We measured erythrocyte creatine in 92 HCM patients. Twelve patients had left ventricular outflow tract obstruction (LVOTO), 4 had midventricular obstruction (MVO), and the remaining 76 were non-obstructive. Erythrocyte creatine levels ranged from 0.92 to 4.36µmol/g hemoglobin. Higher levels of erythrocyte creatine were associated with higher IVPG (r=0.437, p<0.001). If erythrocyte creatine levels are high (≥1.8µmol/g hemoglobin), subclinical hemolysis is considered to be present. Half of LVOTO patients and no MVO patients showed high erythrocyte creatine levels. Although non-obstructive patients did not show significant intraventricular obstruction at rest, some showed high erythrocyte creatine levels. When LVOT-PG was measured during the strain phase of the Valsalva maneuver in 20 non-obstructive patients, 7 of those 20 patients showed LVOTO. In the 20 patients, there was no relation between erythrocyte creatine levels and LVOT-PG before the Valsalva maneuver (r=0.125, p=0.600), whereas there was a significant correlation between erythrocyte creatine and LVOT-PG provoked by the Valsalva maneuver (r=0.695, p=0.001). CONCLUSIONS: There is biochemical evidence of subclinical hemolysis in patients with HCM, and this hemolysis seems to be associated with LVOTO provoked by daily physical activities.